ABSTRACT: Interrogating the cholinergic basis of opioid use disorder Opioids offer unmatched clinical efficacy in the treatment of pain, and offer pronounced therapeutic potential in the treatment of anxiety, depression, and psychosis. Nevertheless opioids come with equally harmful side effects that can lead to opioid use disorder. Three major subtypes of opioid receptors have been identified, which are each expressed in numerous cells. Because traditional drugs impact all cells in a given volume, it has been difficult to map cell type-specific contributions of drug-mediated behavior. To address this gap, we developed DART (drugs acutely restricted by tethering), which works by genetically programming a subset of cells to capture and concentrate a specific drug to levels ~1000 times higher than anywhere else, thus restricting drug action to the chosen subset of cells. Here, we propose to develop, characterize, and distribute a comprehensive toolset focused on opioid neuropharmacology. As a roadmap for the widespread adoption of these reagents, we propose behavioral experiments motivated by a recent double-blind placebo-controlled trial in which a cholinergic drug demonstrated efficacy in the treatment of opioid use disorder. We will test the hypothesis that ?ORs on cholinergic interneurons mediate the harmful (addictive) effects of opioids, independent of helpful (analgesic) effects. The proposed technologies will offer the unprecedented opportunity to establish causal behavioral roles of opioid neuropharmaceuticals mediated by defined cell types. Because the technologies are rooted in therapeutically relevant neuropharmaceuticals, clinical relevance is provided without the need to sacrifice mechanistic rigor.